- Email: [email protected]
Evidence of in situ uptake and incorporation of bicarbonate and amino acids by a hydrothermal vent mussel
J. Exp. Mar. Biol. Ecol., 1986, Vol. 96, pp. 191-198
191
Elsevier
JEM 645
EVIDENCE OF IN SITU UPTAKE AND INCORPORATION OF BICARBONATE AND AMINO ACIDS BY A HYDROTHERMAL VENT MUSSEL
A. FIALA-MEDIONI Laboratoire Arago, University of Paris VI, 66650 Banyuls-sur-Mer. France
A. M. ALAYSE Abyssal Ecology team - IFREMER
Centre de Brest, B.P. 337, 29273 Brest Ctdex, France
and G. CAHET Laboratoire Arago, University of Paris VI, 66650 Banyuls-sur-Mer, France
(Received 15 August 1985; revision received 4 January 1986; accepted 7 January 1986) Abstract: The hydrothermal vent mussel Bathymodiolus sp. is demonstrated to incorporate inorganic CO, from sea water. After N 24 h incubation with HWO; the major part of the radioactivity is incorporated into macromolecules mostly in proteins but also in a notable lipidic fraction. 77 to 98% of this radioactivity is found in the gill and autoradiographs show that CO, fixation is only observed in cells containing high concentrations of bacteria. The results endorse the hypothesis that the associated bacteria might provide a nutritional source for the mussel. The mussel is also able to absorb and incorporate dissolved amino acids. Heterotrophic processes involving dissolved organic matter may interfere with the autotrophic pathways. Beside its capability of feeding on particulate material, the mussel may be thus able to live on reduced carbon and nitrogen compounds synthesized by its associated bacteria as well as on dissolved organic compounds present in sea water. The effective participation of the different processes is probably related to the ecological conditions experienced by the mussel in vent areas. Key words: hydrothermal vent mussel; bacterial association; autotrophic potential; dissolved amino-acid incorporation; Bathymodiolus
INTRODUCTION
The mytilid molluscs in coastal waters are filter-feeders living essentially on particulate matter (Jorgensen, 1966) and possibly partly on dissolved organic material (DOM) (Stephens, 1982). In addition to such possibilities, the mytilid recently discovered associated with deep hydrothermal vents (review in Hessler & Smithey, 1984) may have adapted to obtain part of its energy from chemoautotrophic processes through a symbiotic bacterial association. Indirect evidence of a chemoautotrophic potential in the mussel gill was given by the detection of sulphide-oxidizing enzyme activities (which 0022-0981/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
192
A.FIALA-MkDIONI ETAL.
generate reducing energy and ATP), as well as Calvin-Benson cycle enzymes implicated in the net fixation of CO; (Felbeck et al., 1981). Isotopic analyses demonstrate 13C depletion rates related to an important chemosynthetic carbon fixation (Ray & Hedges, 1979; Williams et al., 1981). Finally, prokaryotic cells were found associated with the more common and abundant organisms of deep-sea hydrothermal vents (Cavanaugh et al., 1981; Felbeck, 1981; Felbeck & Somero, 1982; Desbruyeres et al., 1983) as well as a variety of bivalve molluscs from coastal sulphide-rich areas (Cavanaugh, 1983; Felbeck, 1983; Felbeck et al., 1983; Schweimanns & Felbeck, 1985). All these results led the authors to propose a general scheme of a chemoautotrophic, chemical energybased symbiosis, playing an important role in supplying necessary reduced carbon. Ultrastructural studies have confirmed the presence in the mussel gill of high densities of gram negative bacteria (Fiala-Medioni, 1984; Le Pennec & Hily, 1984) and demonstrated a good integration of the bacteria within the gill cells (Fiala-Medioni, 1984). In situ experiments were carried out from the submersible Cyana during the Biocyarise cruise (EPR, 13 ON at 2600 m depth) to obtain direct evidence of the ability of the mussel to absorb and metabolize radio-labelled bicarbonate and dissolved amino acids. MATERIAL
AND
METHODS
The mussel collected at 13”N belongs to the Bathymodiolus genera but has not yet been confumed to be Bathymodiolus thermophilus described by Kenk & Wilson (1985) in material from Galapagos. Mussels from 12 to 15 cm long were collected with the subsmersible’s claw and placed in 25-l plastic boxes as described by Alayse-Danet et al. (1985). In the first experiment 370 MBq NaH14C0, (2.07 GBq/mM) were injected. In a second experiment a mixture of 370 MBq of amino acids (3H alanine, glycine, proline, leucine, and valine, 1.39 TBq/mM) were injected. A temperature probe was used only in the second experiment and gave 7 and 10 “C near the experimental box. After an incubation time of 23.5 h for both the experiments the boxes were opened, allowing release of radioactive compounds. On board, mussels were immediately dissected, the organs washed in sea water and frozen after the removal and glutaraldehyde futation of small samples for autoradiographic investigations. Organs were then homogenized and scintillation counts made on aliquots in a Beckman scintillator after combustion at 700 “C in a Packard oxidizer. Extractions for biochemical analysis were made according to Wirsen dc Jannasch (1983) and Schlichter et al. (1983) and autoradiograms according to Rogers (1979). For autoradiograms samples of gill lamella were dissected, rinsed in sea water and fixed in 3% glutaraldehyde in a cacodylate buffer 0.4 M at pH 7.8. After dehydratation through propylene oxide, specimens were embedded in Epon 812. Semi-thin sections (x 1 pm) were made and autoradiograms prepared according to Rogers (1979). The slides were dipped in Ilford nuclear emulsion in gel form K5, diluted in 5% water at 43 “C. After 6 days exposure in a refrigerator, they were developed (D19 Kodak l/3
NUTRITION
OF A HYDROTHERMAL
193
VENT MUSSEL
diluted 4 mm), fured, stained with Richardson colouration before being mounted in entellan (Merck). RESULTS UPTAKE
OF 14C LABELLED
Despite could
a certain
be detected
BICARBONATE
variability
AND CO, FIXATION
in the four mussels,
in the mussel
tissues
high concentrations
of radioactivity
after
20 to 24 h incubation with H14CO;. to 93 y0 of this radioactivity was concentrated in the gill (Table I). Autoradiographs a transverse section of a gill lamella clearly show that incorporation does not occur ciliated cells but only in cells containing the associated bacteria. The radioactivity
77 of in is
TABLEI
Distribution ofradioactivity in the different organs after 23.5 h in situ incubation with Hi4CO; : SA, specific activity in dpm . min _ ’ mg dry wt ’ ; RA %, relative activity (obtained from RA = SA x wt of the organ/total wt of organs).
Samples Mussel
1
SA RA% SA RA % SA RA% SA RA%
Mussel 2 Mussel 3 Mussel 4
predominantly concentrated Biochemical found
Digestive tract
Gill
localized (Fig.
1321 86 1185 77 9170 90 8843 93
Mantle
149 il 388 4 907
in the apical zones
106 4 178 I 465 3 226 2
of the cells where
endocellular
Foot and residue 156 9 232 12 804 7 385 5
bacteria
are
of the radioactivity
is
1).
analysis
in small molecules
of the gill demonstrates (amino
acids,
that 41 to 57%
sugars,
etc.)
and that 43
to 59% was incorporated into macromolecules with a predominantly proteic fraction. A notable percentage of incorporation into lipids was also evident (Table II, Fig. 2A). In the mantle, incorporation into macromolecules represented 29 to 46 y0 of the total radioactivity with the dominant fraction (54 to 71%) being found in small molecules (Table II). UPTAKE
AND INCORPORATION
OF 3H AMINO
ACIDS
The results show an evident ability of the mussel to take up amino acids from sea water. After an incubation time of 23.5 h with a 3H amino acid mixture, the different organs of the two mussels used were heavily labelled (Table III). The gill was again the most strongly labelled, containing 48 and 59% of the total radioactivity absorbed. Biochemical tests show an incorporation into macromolecules of 26 and 33 % (Fig. 2B).
A. FIALA-MfiDIONI
ET-AL
A
Fig. 1. Autoradiographs of transverse sections of gill lamella of the hydrothermal vent mussel: A, nonradioactive control; B, sample showing numerous grains (arrows) on the bacterial zone of the ceils (b); L, lipids; scale bar, 100 pm.
Fig. 2. Percentage of radioactivity in the different extracting phases of the gill tissue: 1, small molecules (amino acids and sugars); 2, lipids; 3, proteins and nucleic acids; A, mussel 3 after 23.5 h incubation with H”C0; ; B, mussels 9 and 10 after 23.5 h incubation with 3H amino acids.
NUTRITION
OF A HYDROTHERMAL
195
VENT MUSSEL
TABLE II Percentages of radioactivity in the different extracting phases of gill and mantle after in situ 23.5 h incubation with H14CO; : after treatment with 1, TCA 10% (small molecules: amino acids, sugar, etc.); 2, mixture of 95” ethanol/ether (l/l) (lipids); 3, TCA 10% at 60°C (nucleic acids and polysaccharides); proteins). 4, 1 N NaOH (alcaline soluble proteins); 5, 6 N HCI at 110°C for 24 h (hydrolysable Samples Mussel
1
gill mantle gill mantle gill mantle gill mantle
Mussel 2 Mussel 3 Mussel 4
1
2
3
4
5
45 54 51 66 51 71 41 51
I 12 10 10 15 13 14 13
8 4 5 4 I 4 13 6
38 26 33 14 21 11 31 23
2 4 1 6 1 1 1
TABLE III Distribution of radioactivity in the different organs after 23.5 h in situ incubation with 3H mixture of amino acids: SA, specific activity in dpm min _ ’ mg dry wt - ‘; RA%, percentage of relative activity (see legend Table I).
Samples
Gill
Mussel 9 Mussel
10
SA RA% SA RA%
4204 48 1935 59
Digestive tract 1630 11 1243 8
Mantle 1295 19 1296 9
Foot and residue 1910 22 1261 24
DISCUSSION
The results demonstrate direct biochemical and autoradiographic evidence for CO, fxation in the mussel tissues. They confii the gill of the mussel to be the site of biosynthesis of compounds derived from inorganic CO,. The incorporation of radioactivity from bicarbonate into metabolites has been observed in some invertebrate tissues in the absence of autotrophy (Hammen & Osborne, 1959). These carboxylation processes do not seem to interfere in our experiments as the incorporation of labelled bicarbonate is not observed in ciliated cells but only in the bacterial zone of the cells containing the associated bacteria. This result is in favour of the chemoautotrophic potential of the bacteria previously indirectly demonstrated by the detection of Calvin-Benson cycle enzymes in the gill tissue of the mussel (Felbeck et al., 1981). Some variability exists in the relative radioactivity concentrations in different mussels from the same experiment. This variability is probably related to differences in the actual contact time of the gill with the labelled water, since all the mussels may not have opened their valves and begun to pump at the same time. But possible differences in the
196
A. FIALA-MfiDIONI ETAL
experimental temperature in the boxes may also have had an effect on the rate of incorporation. The difficulty in determining precisely, in the in situ conditions, when contact between the gill and labelled sea water was made, does not permit us to calculate incorporation rates. In the gill, 7 to 15% dry mass of the radioactivity has been found incorporated in the lipidic fraction. This result might explain the accumulation of lipids revealed under the bacterial zone of the contaminated gill cells by TEM observations (Fiala-Medioni, 1984; Fiala-Medioni et al., in press). The specific activity found in lipids (7 to 15% dry mass) and proteins (21 to 40% dry mass) show changes when compared with the concentrations of lipids (5.2 to 6.2% dry mass) and proteins (51 to 59.7% dry mass) found by Smith (1985) in Bathymodiolus thermophilus of the Galapagos. This may be related to differences in the metabolic activity of the associated bacteria. Biochemical evidence of CO, fixation has been reported for a number of other species of bivalves living in coastal sulphide-rich mud which have also developed a symbiotic relationship with gram negative bacteria (Cavanaugh, 1983; Felbeck, 1983). This fixation is enhanced by the presence of sulphur compounds (Cavanaugh, 1983). The ability of these species to oxidize HS - and produce SO; shows that the symbiotic bacteria are capable of converting HS - to oxidized sulphur compounds (Felbeck, 1983). The blood of Calyptogena magnifca, another bivalve associated with hydrothermal vents and containing bacteria in gill cells (Cavanaugh, 1983; Fiala-Medioni, 1984), has been found to contain high levels of sulphide and a binding protein which may mediate the transport of sulphide from the environment to the symbionts (Arp et al., 1984). The demonstration of this sulphide chemosynthetic system has led the authors to conclude that symbiotically generated nutrients may be the most significant source of energy for this species (Felbeck & Somero, 1982; Cavanaugh, 1983; Felbeck, 1983). Our results endorse the hypothesis that this energetic source may be also used by this hydrothermal vent mussel. Uptake of DOM, however, may be another source of reduced carbon for the mussel’s nutrition. In terms of energetic efficiency it might be more economic for the mussel to get energy from heterotrophic processes involving DOM. Our results show that this possibility is certainly fully exploited by the vent mytilid in the same manner as littoral ones (Stephens, 1982; Jorgensen, 1983). In the absence of data on hydrothermal vent amino-acid concentrations and in order not to increase signiticantly the natural concentrations in our experiments we used concentrations in the range of values obtained in offshore and deep waters (review in Stephens, 1982). Yet the important biological production of these zones (Hessler & Smithey, 1984; Desbruyeres & Laubier, 1984) and especially by chemosynthetic bacteria (Jannasch & Wirsen, 1979; Karl et al., 1980; Ruby et al., 1981) suggest higher concentrations. Recent results on the 13”N site show much higher fatty acid concentrations in the vent areas than in waters away from the vents but at the same depth (Bra&, 1984). These results also demonstrate a great variability in the spatial distribution of the organic material possibly in relation to convective micro-currents.
NUTRITION OF A HYDROTHERMAL
VENT MUSSEL
197
The possibility of using DOM associated with the ability of the mussel to exploit the suspended particulate material as attested by the digestive tract anatomy as well as gut content analyses (Le Pennec & Prieur, 1984) may explain the ecological distribution of this mussel which can survive away from the direct flux of vent water (Hessler & Smithey, 1984; Desbruyeres & Laubier, 1984). It is hypothesized that a possible balance between autotrophic and heterotrophic processes may be established in relation to the relative concentrations in HS -, DOM, and particulate material (which probably greatly vary in space and time) around the organisms.
ACKNOWLEDGEMENTS
This work was supported by the IFREMER grant No 84.7635 and CNRS UA.117. Professor L. Laubier is acknowledged for a critical revision of the manuscript and R. Tait for the correction of the English. We are also indebted to D. Desbruyeres for facilities during the Biocyarise cruise, to P. Crassous, A. Echardour, and the crew of the submersible for excellent technical assistance. The semi-thin sections for autoradiographs were made by N. Mosconi.
REFERENCES ALAYSE-DANET,A.M., F. GAILL & D. DESBRUY~RES,1985. Preliminary studies on the relationship between the Pompei worm, Alvinella pompejana (Polychaeta : Ampharetidae), and its epibiotic bacteria. Proc. 19th Eur. Mar. Biol. Symp., edited by P.E. Gibbs, Cambridge University Press, Cambridge, pp. 167-172. ARP, A. J., J. J. CHILDRESS& C. R. FISHER, 1984. Metabolic and blood gas transport characteristics of the hydrothermal vent bivalve Calyptogena magnifca. Physiol. Zool., Vol. 57, pp. 648-662. BRAULT,M., 1984. Biogeochimie de la mat&e organique dans les environnements hydrothermaux le long de la dorsale est Pacitique a 13”N. These de 3eme cycle, Universite P.M. Curie (Paris 6), 168 pp. CAVANAUGH,C. M., 1983. Symbiotic chemoautotrophic bacteriain marine invertebrates from sulphide-rich habitats. Nature (London), Vol. 302, pp. 58-61. CAVANAUGH,CM., S.L. GARDINER, M.L. JONES, M.W. JANNASCH & J.R. WATERBURY,1981. Prokaryotic cells in the hydrothermal vent tube worm Riitiapachyptila Jones: possible chemoautotrophic symbionts. Science, Vol. 213, pp. 340-341. DESBRUY~RES,D., F. GAILL, L. LAUBIER,D. PRIEUR& G. H. RAU, 1983. Unusual nutrition ofthe “Pompei worm” Alvinellapompejana (polychaetous amrelid) from a hydrothermal vent environment: SEM, TEM, Cl3 and N15 evidence. Mar. Biol., Vol. 75, pp. 201-205. DESBRUY~RES,D. & L. LAUBIER,1984. Primary consumers from hydrothermal vent animal communities. In, Hydrothermal processes at seafloor spreading centers, edited by P. A. Rona et al., Plenum, New York, pp. 7 1l-734. FELBECK,H., 1981. Chemoautotrophic potential ofthe hydrothermal vent tube worm Rijliapachyptila Jones (Vestimentifera). Science, Vol. 213, pp. 336-338. FELBECK,H., 1983. Sulfide oxydation and carbon fmation by the gutless clam Solemya reidi: an animalbacteria symbiosis. J. Comp. Physiol., Vol. 152, pp. 3-l 1. FELBECK,H., J. J. CHILDRESS& G.N. SOMERO,1981. Calvin-Benson cycle and sulfide oxydation enzymes in animals from sulphide-rich habitats. Nature (London), Vol. 293, pp. 291-293. FELBECK,H., J. J. CH~LDRESS& G.N. SOMERO,1983. Biochemical interactions between molluscs and their algal and bacterial symbionts. In, The Mollusca, edited by K.M. Wilbur, Academic Press, New York, pp. 331-358.
198
A. FIALA-MEDIONI
ETAL.
FELBECK,H. & G. H. SOMERO,1982. Primary production in deep-sea hydrothermal vent organisms: roles of sulfide-oxidizing bacteria. Trends Biochem Sci., Vol. 7, pp. 201-204. FIALA-MBDIONI,A., 1984. Ultrastructural evidence of abundance of intracellular symbiotic bacteria in the gill of bivalve molluscs of deep hydrothermal vents. C.R. Acad. Sci. Paris, Ser. III, Vol. 298, pp. 487-492. FIALA-MEDIONI,A., C. METIVIER,A. HERRY& M. LE PENNEC,in press. Ultrastructure of the gill filament of an hydrothermal vent mytilid. Mar. Biol. HAMMEN,C. S. & P.J. OSBORNE,1959. Carbon dioxide fixation in marine invertebrates: a survey of major phyla. Science, Vol. 130, pp. 1409-1410. HESSLER, R. R. & W. M. SMITHEY,1984. The distribution and community structure of megafauna at the Galapagos rift hydrothermal vents. In, Hydrothermalprocesses at seafloor spreading center, edited by P. A. Rona et al., Plenum, New York, pp. 735-770. JANNASCH,H.W. & CO. WIRSEN, 1979. Chemosynthetic primary production at east Pacific sea-floor spreading center. BioScience, Vol. 29, pp. 592-598. JBRGENSEN,C.B., 1966. Biology of suspension feeding, edited by C. A. Kerkut, Pergamon Press, Oxford, 357 pp. JBRGENSEN,C. B., 1983. Patterns ofuptake of dissolved amino-acids in mussels (Mytilus edulis). Mar. Biol., Vol. 73, pp. 177-182. KARL, D., C.O. WIRSEN & H.W. JANNASCH, 1980. Deep-sea primary production at the Galapagos hydrothermal vents. Science, Vol. 207, pp. 1345-1347. KENK, V. D. & B. R. WILSON, 1985. A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos rift zone. Malacologia, Vol. 26, pp. 253-211. LE PENNEC,M. & A. HILY, 1984. Anatomie, structure et ultrastructure de la branchie dun Mytilidae des sites hydrothermaux du Pacifique oriental. Acta Oceanol., Vol. 7, pp. 517-523. LE PENNEC,M. & D. PRIEUR, 1984. Observations sur la nutrition dun Mytilidae dun site hydrothermal actif de la dorsale du Pacitique Oriental. CR. Acad. Sci. Paris, Vol. 298, pp. 493-498. RAY, G.H. & J.I. HEDGES, 1979. Carbon 13 depletion in a hydrothermal vent mussel: suggestion of a chemosynthetic food source. Science, Vol. 203, pp. 648-649. ROGERS, A. W., 1979. Techniques of autoradiography. Elsevier, Amsterdam, 3rd edition, 372 pp. RUBY,E.G., C. 0. WIRSEN& H. W. JANNASCH,1981. Chemolithotrophic sulfur-oxidizing bacteria from the Galapagos rift hydrothermal vents. Appl. Environ. Microbial., Vol. 42, pp. 317-324. SCHLICHTER,D. S., A. SVOBODA& B. P. KREMER, 1983. Functional autotrophy of Heteroxeniafuscecens (Anthozoa : Alcyonaria): carbon assimilation and translation of photosynthates from symbionts to host. Mar. Biol., Vol. 78, pp. 29-38. SCHWEIMANNS,M. & H. FELBECK, 1985. Significance of the occurrence of chemoautotrophic bacterial endosymbionts in lucinid clams from Bermuda. Mar. Ecol. Prog. Ser., Vol. 24, pp. 113-120. SMITH,K.L., 1985. Deep-sea hydrothermal vent mussel: nutritional state and distribution at the Galapagos rift. Ecology, Vol. 66, pp. 1067-1080. STEPHENS,G. C., 1982. The trophic role of dissolved organic material. In, Analysis of marine ecosystems, edited by A.L. Longhurst, Academic Press, New York, pp. 271-291. WILLIAMS,P.M., K.L. SMITH, R.M. DRUFFEL & T.W. LINICK, 1981. Dietary carbon sources of mussels and tube worms from Galapagos hydrothermal vents determined from tissue Cl4 activity. Nature (London), Vol. 292, pp. 448-449. WIRSEN, C.O. & H.W. JANNASCH, 1983. In situ studies on deep-sea amphipods and their intestinal microflora. Mar. Biol., Vol. 78, pp. 69-73.
191
Elsevier
JEM 645
EVIDENCE OF IN SITU UPTAKE AND INCORPORATION OF BICARBONATE AND AMINO ACIDS BY A HYDROTHERMAL VENT MUSSEL
A. FIALA-MEDIONI Laboratoire Arago, University of Paris VI, 66650 Banyuls-sur-Mer. France
A. M. ALAYSE Abyssal Ecology team - IFREMER
Centre de Brest, B.P. 337, 29273 Brest Ctdex, France
and G. CAHET Laboratoire Arago, University of Paris VI, 66650 Banyuls-sur-Mer, France
(Received 15 August 1985; revision received 4 January 1986; accepted 7 January 1986) Abstract: The hydrothermal vent mussel Bathymodiolus sp. is demonstrated to incorporate inorganic CO, from sea water. After N 24 h incubation with HWO; the major part of the radioactivity is incorporated into macromolecules mostly in proteins but also in a notable lipidic fraction. 77 to 98% of this radioactivity is found in the gill and autoradiographs show that CO, fixation is only observed in cells containing high concentrations of bacteria. The results endorse the hypothesis that the associated bacteria might provide a nutritional source for the mussel. The mussel is also able to absorb and incorporate dissolved amino acids. Heterotrophic processes involving dissolved organic matter may interfere with the autotrophic pathways. Beside its capability of feeding on particulate material, the mussel may be thus able to live on reduced carbon and nitrogen compounds synthesized by its associated bacteria as well as on dissolved organic compounds present in sea water. The effective participation of the different processes is probably related to the ecological conditions experienced by the mussel in vent areas. Key words: hydrothermal vent mussel; bacterial association; autotrophic potential; dissolved amino-acid incorporation; Bathymodiolus
INTRODUCTION
The mytilid molluscs in coastal waters are filter-feeders living essentially on particulate matter (Jorgensen, 1966) and possibly partly on dissolved organic material (DOM) (Stephens, 1982). In addition to such possibilities, the mytilid recently discovered associated with deep hydrothermal vents (review in Hessler & Smithey, 1984) may have adapted to obtain part of its energy from chemoautotrophic processes through a symbiotic bacterial association. Indirect evidence of a chemoautotrophic potential in the mussel gill was given by the detection of sulphide-oxidizing enzyme activities (which 0022-0981/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
192
A.FIALA-MkDIONI ETAL.
generate reducing energy and ATP), as well as Calvin-Benson cycle enzymes implicated in the net fixation of CO; (Felbeck et al., 1981). Isotopic analyses demonstrate 13C depletion rates related to an important chemosynthetic carbon fixation (Ray & Hedges, 1979; Williams et al., 1981). Finally, prokaryotic cells were found associated with the more common and abundant organisms of deep-sea hydrothermal vents (Cavanaugh et al., 1981; Felbeck, 1981; Felbeck & Somero, 1982; Desbruyeres et al., 1983) as well as a variety of bivalve molluscs from coastal sulphide-rich areas (Cavanaugh, 1983; Felbeck, 1983; Felbeck et al., 1983; Schweimanns & Felbeck, 1985). All these results led the authors to propose a general scheme of a chemoautotrophic, chemical energybased symbiosis, playing an important role in supplying necessary reduced carbon. Ultrastructural studies have confirmed the presence in the mussel gill of high densities of gram negative bacteria (Fiala-Medioni, 1984; Le Pennec & Hily, 1984) and demonstrated a good integration of the bacteria within the gill cells (Fiala-Medioni, 1984). In situ experiments were carried out from the submersible Cyana during the Biocyarise cruise (EPR, 13 ON at 2600 m depth) to obtain direct evidence of the ability of the mussel to absorb and metabolize radio-labelled bicarbonate and dissolved amino acids. MATERIAL
AND
METHODS
The mussel collected at 13”N belongs to the Bathymodiolus genera but has not yet been confumed to be Bathymodiolus thermophilus described by Kenk & Wilson (1985) in material from Galapagos. Mussels from 12 to 15 cm long were collected with the subsmersible’s claw and placed in 25-l plastic boxes as described by Alayse-Danet et al. (1985). In the first experiment 370 MBq NaH14C0, (2.07 GBq/mM) were injected. In a second experiment a mixture of 370 MBq of amino acids (3H alanine, glycine, proline, leucine, and valine, 1.39 TBq/mM) were injected. A temperature probe was used only in the second experiment and gave 7 and 10 “C near the experimental box. After an incubation time of 23.5 h for both the experiments the boxes were opened, allowing release of radioactive compounds. On board, mussels were immediately dissected, the organs washed in sea water and frozen after the removal and glutaraldehyde futation of small samples for autoradiographic investigations. Organs were then homogenized and scintillation counts made on aliquots in a Beckman scintillator after combustion at 700 “C in a Packard oxidizer. Extractions for biochemical analysis were made according to Wirsen dc Jannasch (1983) and Schlichter et al. (1983) and autoradiograms according to Rogers (1979). For autoradiograms samples of gill lamella were dissected, rinsed in sea water and fixed in 3% glutaraldehyde in a cacodylate buffer 0.4 M at pH 7.8. After dehydratation through propylene oxide, specimens were embedded in Epon 812. Semi-thin sections (x 1 pm) were made and autoradiograms prepared according to Rogers (1979). The slides were dipped in Ilford nuclear emulsion in gel form K5, diluted in 5% water at 43 “C. After 6 days exposure in a refrigerator, they were developed (D19 Kodak l/3
NUTRITION
OF A HYDROTHERMAL
193
VENT MUSSEL
diluted 4 mm), fured, stained with Richardson colouration before being mounted in entellan (Merck). RESULTS UPTAKE
OF 14C LABELLED
Despite could
a certain
be detected
BICARBONATE
variability
AND CO, FIXATION
in the four mussels,
in the mussel
tissues
high concentrations
of radioactivity
after
20 to 24 h incubation with H14CO;. to 93 y0 of this radioactivity was concentrated in the gill (Table I). Autoradiographs a transverse section of a gill lamella clearly show that incorporation does not occur ciliated cells but only in cells containing the associated bacteria. The radioactivity
77 of in is
TABLEI
Distribution ofradioactivity in the different organs after 23.5 h in situ incubation with Hi4CO; : SA, specific activity in dpm . min _ ’ mg dry wt ’ ; RA %, relative activity (obtained from RA = SA x wt of the organ/total wt of organs).
Samples Mussel
1
SA RA% SA RA % SA RA% SA RA%
Mussel 2 Mussel 3 Mussel 4
predominantly concentrated Biochemical found
Digestive tract
Gill
localized (Fig.
1321 86 1185 77 9170 90 8843 93
Mantle
149 il 388 4 907
in the apical zones
106 4 178 I 465 3 226 2
of the cells where
endocellular
Foot and residue 156 9 232 12 804 7 385 5
bacteria
are
of the radioactivity
is
1).
analysis
in small molecules
of the gill demonstrates (amino
acids,
that 41 to 57%
sugars,
etc.)
and that 43
to 59% was incorporated into macromolecules with a predominantly proteic fraction. A notable percentage of incorporation into lipids was also evident (Table II, Fig. 2A). In the mantle, incorporation into macromolecules represented 29 to 46 y0 of the total radioactivity with the dominant fraction (54 to 71%) being found in small molecules (Table II). UPTAKE
AND INCORPORATION
OF 3H AMINO
ACIDS
The results show an evident ability of the mussel to take up amino acids from sea water. After an incubation time of 23.5 h with a 3H amino acid mixture, the different organs of the two mussels used were heavily labelled (Table III). The gill was again the most strongly labelled, containing 48 and 59% of the total radioactivity absorbed. Biochemical tests show an incorporation into macromolecules of 26 and 33 % (Fig. 2B).
A. FIALA-MfiDIONI
ET-AL
A
Fig. 1. Autoradiographs of transverse sections of gill lamella of the hydrothermal vent mussel: A, nonradioactive control; B, sample showing numerous grains (arrows) on the bacterial zone of the ceils (b); L, lipids; scale bar, 100 pm.
Fig. 2. Percentage of radioactivity in the different extracting phases of the gill tissue: 1, small molecules (amino acids and sugars); 2, lipids; 3, proteins and nucleic acids; A, mussel 3 after 23.5 h incubation with H”C0; ; B, mussels 9 and 10 after 23.5 h incubation with 3H amino acids.
NUTRITION
OF A HYDROTHERMAL
195
VENT MUSSEL
TABLE II Percentages of radioactivity in the different extracting phases of gill and mantle after in situ 23.5 h incubation with H14CO; : after treatment with 1, TCA 10% (small molecules: amino acids, sugar, etc.); 2, mixture of 95” ethanol/ether (l/l) (lipids); 3, TCA 10% at 60°C (nucleic acids and polysaccharides); proteins). 4, 1 N NaOH (alcaline soluble proteins); 5, 6 N HCI at 110°C for 24 h (hydrolysable Samples Mussel
1
gill mantle gill mantle gill mantle gill mantle
Mussel 2 Mussel 3 Mussel 4
1
2
3
4
5
45 54 51 66 51 71 41 51
I 12 10 10 15 13 14 13
8 4 5 4 I 4 13 6
38 26 33 14 21 11 31 23
2 4 1 6 1 1 1
TABLE III Distribution of radioactivity in the different organs after 23.5 h in situ incubation with 3H mixture of amino acids: SA, specific activity in dpm min _ ’ mg dry wt - ‘; RA%, percentage of relative activity (see legend Table I).
Samples
Gill
Mussel 9 Mussel
10
SA RA% SA RA%
4204 48 1935 59
Digestive tract 1630 11 1243 8
Mantle 1295 19 1296 9
Foot and residue 1910 22 1261 24
DISCUSSION
The results demonstrate direct biochemical and autoradiographic evidence for CO, fxation in the mussel tissues. They confii the gill of the mussel to be the site of biosynthesis of compounds derived from inorganic CO,. The incorporation of radioactivity from bicarbonate into metabolites has been observed in some invertebrate tissues in the absence of autotrophy (Hammen & Osborne, 1959). These carboxylation processes do not seem to interfere in our experiments as the incorporation of labelled bicarbonate is not observed in ciliated cells but only in the bacterial zone of the cells containing the associated bacteria. This result is in favour of the chemoautotrophic potential of the bacteria previously indirectly demonstrated by the detection of Calvin-Benson cycle enzymes in the gill tissue of the mussel (Felbeck et al., 1981). Some variability exists in the relative radioactivity concentrations in different mussels from the same experiment. This variability is probably related to differences in the actual contact time of the gill with the labelled water, since all the mussels may not have opened their valves and begun to pump at the same time. But possible differences in the
196
A. FIALA-MfiDIONI ETAL
experimental temperature in the boxes may also have had an effect on the rate of incorporation. The difficulty in determining precisely, in the in situ conditions, when contact between the gill and labelled sea water was made, does not permit us to calculate incorporation rates. In the gill, 7 to 15% dry mass of the radioactivity has been found incorporated in the lipidic fraction. This result might explain the accumulation of lipids revealed under the bacterial zone of the contaminated gill cells by TEM observations (Fiala-Medioni, 1984; Fiala-Medioni et al., in press). The specific activity found in lipids (7 to 15% dry mass) and proteins (21 to 40% dry mass) show changes when compared with the concentrations of lipids (5.2 to 6.2% dry mass) and proteins (51 to 59.7% dry mass) found by Smith (1985) in Bathymodiolus thermophilus of the Galapagos. This may be related to differences in the metabolic activity of the associated bacteria. Biochemical evidence of CO, fixation has been reported for a number of other species of bivalves living in coastal sulphide-rich mud which have also developed a symbiotic relationship with gram negative bacteria (Cavanaugh, 1983; Felbeck, 1983). This fixation is enhanced by the presence of sulphur compounds (Cavanaugh, 1983). The ability of these species to oxidize HS - and produce SO; shows that the symbiotic bacteria are capable of converting HS - to oxidized sulphur compounds (Felbeck, 1983). The blood of Calyptogena magnifca, another bivalve associated with hydrothermal vents and containing bacteria in gill cells (Cavanaugh, 1983; Fiala-Medioni, 1984), has been found to contain high levels of sulphide and a binding protein which may mediate the transport of sulphide from the environment to the symbionts (Arp et al., 1984). The demonstration of this sulphide chemosynthetic system has led the authors to conclude that symbiotically generated nutrients may be the most significant source of energy for this species (Felbeck & Somero, 1982; Cavanaugh, 1983; Felbeck, 1983). Our results endorse the hypothesis that this energetic source may be also used by this hydrothermal vent mussel. Uptake of DOM, however, may be another source of reduced carbon for the mussel’s nutrition. In terms of energetic efficiency it might be more economic for the mussel to get energy from heterotrophic processes involving DOM. Our results show that this possibility is certainly fully exploited by the vent mytilid in the same manner as littoral ones (Stephens, 1982; Jorgensen, 1983). In the absence of data on hydrothermal vent amino-acid concentrations and in order not to increase signiticantly the natural concentrations in our experiments we used concentrations in the range of values obtained in offshore and deep waters (review in Stephens, 1982). Yet the important biological production of these zones (Hessler & Smithey, 1984; Desbruyeres & Laubier, 1984) and especially by chemosynthetic bacteria (Jannasch & Wirsen, 1979; Karl et al., 1980; Ruby et al., 1981) suggest higher concentrations. Recent results on the 13”N site show much higher fatty acid concentrations in the vent areas than in waters away from the vents but at the same depth (Bra&, 1984). These results also demonstrate a great variability in the spatial distribution of the organic material possibly in relation to convective micro-currents.
NUTRITION OF A HYDROTHERMAL
VENT MUSSEL
197
The possibility of using DOM associated with the ability of the mussel to exploit the suspended particulate material as attested by the digestive tract anatomy as well as gut content analyses (Le Pennec & Prieur, 1984) may explain the ecological distribution of this mussel which can survive away from the direct flux of vent water (Hessler & Smithey, 1984; Desbruyeres & Laubier, 1984). It is hypothesized that a possible balance between autotrophic and heterotrophic processes may be established in relation to the relative concentrations in HS -, DOM, and particulate material (which probably greatly vary in space and time) around the organisms.
ACKNOWLEDGEMENTS
This work was supported by the IFREMER grant No 84.7635 and CNRS UA.117. Professor L. Laubier is acknowledged for a critical revision of the manuscript and R. Tait for the correction of the English. We are also indebted to D. Desbruyeres for facilities during the Biocyarise cruise, to P. Crassous, A. Echardour, and the crew of the submersible for excellent technical assistance. The semi-thin sections for autoradiographs were made by N. Mosconi.
REFERENCES ALAYSE-DANET,A.M., F. GAILL & D. DESBRUY~RES,1985. Preliminary studies on the relationship between the Pompei worm, Alvinella pompejana (Polychaeta : Ampharetidae), and its epibiotic bacteria. Proc. 19th Eur. Mar. Biol. Symp., edited by P.E. Gibbs, Cambridge University Press, Cambridge, pp. 167-172. ARP, A. J., J. J. CHILDRESS& C. R. FISHER, 1984. Metabolic and blood gas transport characteristics of the hydrothermal vent bivalve Calyptogena magnifca. Physiol. Zool., Vol. 57, pp. 648-662. BRAULT,M., 1984. Biogeochimie de la mat&e organique dans les environnements hydrothermaux le long de la dorsale est Pacitique a 13”N. These de 3eme cycle, Universite P.M. Curie (Paris 6), 168 pp. CAVANAUGH,C. M., 1983. Symbiotic chemoautotrophic bacteriain marine invertebrates from sulphide-rich habitats. Nature (London), Vol. 302, pp. 58-61. CAVANAUGH,CM., S.L. GARDINER, M.L. JONES, M.W. JANNASCH & J.R. WATERBURY,1981. Prokaryotic cells in the hydrothermal vent tube worm Riitiapachyptila Jones: possible chemoautotrophic symbionts. Science, Vol. 213, pp. 340-341. DESBRUY~RES,D., F. GAILL, L. LAUBIER,D. PRIEUR& G. H. RAU, 1983. Unusual nutrition ofthe “Pompei worm” Alvinellapompejana (polychaetous amrelid) from a hydrothermal vent environment: SEM, TEM, Cl3 and N15 evidence. Mar. Biol., Vol. 75, pp. 201-205. DESBRUY~RES,D. & L. LAUBIER,1984. Primary consumers from hydrothermal vent animal communities. In, Hydrothermal processes at seafloor spreading centers, edited by P. A. Rona et al., Plenum, New York, pp. 7 1l-734. FELBECK,H., 1981. Chemoautotrophic potential ofthe hydrothermal vent tube worm Rijliapachyptila Jones (Vestimentifera). Science, Vol. 213, pp. 336-338. FELBECK,H., 1983. Sulfide oxydation and carbon fmation by the gutless clam Solemya reidi: an animalbacteria symbiosis. J. Comp. Physiol., Vol. 152, pp. 3-l 1. FELBECK,H., J. J. CHILDRESS& G.N. SOMERO,1981. Calvin-Benson cycle and sulfide oxydation enzymes in animals from sulphide-rich habitats. Nature (London), Vol. 293, pp. 291-293. FELBECK,H., J. J. CH~LDRESS& G.N. SOMERO,1983. Biochemical interactions between molluscs and their algal and bacterial symbionts. In, The Mollusca, edited by K.M. Wilbur, Academic Press, New York, pp. 331-358.
198
A. FIALA-MEDIONI
ETAL.
FELBECK,H. & G. H. SOMERO,1982. Primary production in deep-sea hydrothermal vent organisms: roles of sulfide-oxidizing bacteria. Trends Biochem Sci., Vol. 7, pp. 201-204. FIALA-MBDIONI,A., 1984. Ultrastructural evidence of abundance of intracellular symbiotic bacteria in the gill of bivalve molluscs of deep hydrothermal vents. C.R. Acad. Sci. Paris, Ser. III, Vol. 298, pp. 487-492. FIALA-MEDIONI,A., C. METIVIER,A. HERRY& M. LE PENNEC,in press. Ultrastructure of the gill filament of an hydrothermal vent mytilid. Mar. Biol. HAMMEN,C. S. & P.J. OSBORNE,1959. Carbon dioxide fixation in marine invertebrates: a survey of major phyla. Science, Vol. 130, pp. 1409-1410. HESSLER, R. R. & W. M. SMITHEY,1984. The distribution and community structure of megafauna at the Galapagos rift hydrothermal vents. In, Hydrothermalprocesses at seafloor spreading center, edited by P. A. Rona et al., Plenum, New York, pp. 735-770. JANNASCH,H.W. & CO. WIRSEN, 1979. Chemosynthetic primary production at east Pacific sea-floor spreading center. BioScience, Vol. 29, pp. 592-598. JBRGENSEN,C.B., 1966. Biology of suspension feeding, edited by C. A. Kerkut, Pergamon Press, Oxford, 357 pp. JBRGENSEN,C. B., 1983. Patterns ofuptake of dissolved amino-acids in mussels (Mytilus edulis). Mar. Biol., Vol. 73, pp. 177-182. KARL, D., C.O. WIRSEN & H.W. JANNASCH, 1980. Deep-sea primary production at the Galapagos hydrothermal vents. Science, Vol. 207, pp. 1345-1347. KENK, V. D. & B. R. WILSON, 1985. A new mussel (Bivalvia, Mytilidae) from hydrothermal vents in the Galapagos rift zone. Malacologia, Vol. 26, pp. 253-211. LE PENNEC,M. & A. HILY, 1984. Anatomie, structure et ultrastructure de la branchie dun Mytilidae des sites hydrothermaux du Pacifique oriental. Acta Oceanol., Vol. 7, pp. 517-523. LE PENNEC,M. & D. PRIEUR, 1984. Observations sur la nutrition dun Mytilidae dun site hydrothermal actif de la dorsale du Pacitique Oriental. CR. Acad. Sci. Paris, Vol. 298, pp. 493-498. RAY, G.H. & J.I. HEDGES, 1979. Carbon 13 depletion in a hydrothermal vent mussel: suggestion of a chemosynthetic food source. Science, Vol. 203, pp. 648-649. ROGERS, A. W., 1979. Techniques of autoradiography. Elsevier, Amsterdam, 3rd edition, 372 pp. RUBY,E.G., C. 0. WIRSEN& H. W. JANNASCH,1981. Chemolithotrophic sulfur-oxidizing bacteria from the Galapagos rift hydrothermal vents. Appl. Environ. Microbial., Vol. 42, pp. 317-324. SCHLICHTER,D. S., A. SVOBODA& B. P. KREMER, 1983. Functional autotrophy of Heteroxeniafuscecens (Anthozoa : Alcyonaria): carbon assimilation and translation of photosynthates from symbionts to host. Mar. Biol., Vol. 78, pp. 29-38. SCHWEIMANNS,M. & H. FELBECK, 1985. Significance of the occurrence of chemoautotrophic bacterial endosymbionts in lucinid clams from Bermuda. Mar. Ecol. Prog. Ser., Vol. 24, pp. 113-120. SMITH,K.L., 1985. Deep-sea hydrothermal vent mussel: nutritional state and distribution at the Galapagos rift. Ecology, Vol. 66, pp. 1067-1080. STEPHENS,G. C., 1982. The trophic role of dissolved organic material. In, Analysis of marine ecosystems, edited by A.L. Longhurst, Academic Press, New York, pp. 271-291. WILLIAMS,P.M., K.L. SMITH, R.M. DRUFFEL & T.W. LINICK, 1981. Dietary carbon sources of mussels and tube worms from Galapagos hydrothermal vents determined from tissue Cl4 activity. Nature (London), Vol. 292, pp. 448-449. WIRSEN, C.O. & H.W. JANNASCH, 1983. In situ studies on deep-sea amphipods and their intestinal microflora. Mar. Biol., Vol. 78, pp. 69-73.